Cleavable surfactants and methods of use thereof

ABSTRACT

Cleavable compositions and methods of use especially in MALDI MS analysis of hydrophobic proteins.

This application is a 371 of PCT/US02/16640 filed May 28, 2002 whichclaims benefit of U.S. 60/294,337 filed May 29, 2001.

FIELD OF THE INVENTION

The present invention relates generally to cleavable detergents orsurfactants and methods of use thereof including sample isolation,solubilization, emulsification, and analysis. Furthermore, the presentinvention relates to cleavable surfactants which are useful for samplepreparation, but which can be cleaved for removal or to yield cleavageproducts which have additional useful properties, including for matrixassisted laser desorption ionization mass spectroscopy (MALDI MS)analysis of hydrophobic molecules including natural and syntheticpolymers and polypeptides.

The cleavable detergents or surfactants of the present invention, amongother things in additional embodiments, improve the quality of MALDI MSanalyses of proteins, including high molecular weight proteinsassociated with biological tissue.

BACKGROUND OF THE INVENTION

Proteomics, the study of proteins and their functions, is currently afocus of both university and commercial investment as each discovery inproteomics holds the potential to unlock yet another advance in medicalscience. The extreme variability in the chemistry of proteins inbiological systems and especially in mammals, presents special problems.

A recurring problem with respect to proteomics involves the poorsolubility of a large percentage of proteins such as those found inlipid membranes and other hydrophobic areas of the cell or in thecellular environment. This is because many of the systems developed forthe study of proteins are geared to analysis in an aqueous environment.To isolate hydrophobic proteins or hydrophobic protein domains,surfactants (detergents, such as sodium dodecyl sulfate (SDS) or tritonX) are commonly employed. Surfactants generally have a polar head groupand a hydrophobic tail group and encapsulate hydrophobic proteinswherein the hydrophobic tail is in contact with the hydrophobic proteinand the polar groups are in contact with the water. Thus, hydrophobicproteins and polypeptides are sequestered in a coating of detergentwherein the complex is soluble in an aqueous environment.

However, many analytical systems are sensitive to the presence ofsurfactants. For example, SDS and triton X suppress the analyte signalduring matrix assisted laser desorption ionization mass spectrometry(MALDI MS) analysis. Signal suppression from surfactant contamination iscontemplated to result from physical and chemical blockage of theionization/desorption process of MALDI MS.

What is needed are surfactant compositions and methods suitable forMALDI-MS analyses, and other analyses, of hydrophobic moleculesincluding natural and synthetic polymers and polypeptides/proteins.

International Publication WO 00/70334 to Lee et al., discloses certainsurfactants and results for electrospray mass spectroscopy (MS) analysisof myoglobin in the presence of certain of the surfactants.

U.S. Pat. No. 4,713,486 to Buckle discloses certain arachidonic acidanalogues, including certain cinnamates, stated to be useful in thetreatment of allergic diseases.

U.S. Pat. No. 5,114,851 to Porter et al., discloses certain lightactivated acyl-enzymes.

U.S. Pat. No. 5,218,187 to Porter et al., discloses certain compoundsuseful as an intermediate for making light-activatable acyl-enzymes.

Also see U.S. Pat. No. 5,808,300 to Caprioli, incorporated herein byreference, for a discussion of MALDI MS.

Additional background information may be found in the followingpublications: Kyte et al., J. Mol. Biol. (1982) 157(1):105–32; March'sAdvanced Organic Chemistry Reactions, Mechanisms, and Structure, 5^(th)Ed. by Michael B. Smith and Jerry March, John Wiley & Sons, Publishers;Wuts et al. (1999) Protective Groups in Organic Synthesis, 3^(rd) Ed.,John Wiley & Sons, Publishers; Behforouz, M.; Kerwood, J. E. Alkyl andAryl Sulfenimides. J. Org. Chem., 34 (1), 51–55 (1969); and Harpp, D.N.; Ash, D. K.; Back, T. G.; Gleason, J. G.; Orwig, B. A.; VanHorn, W.F. A New Synthesis of Unsymmetrical Disulifdes. Tetrahedron Letters, 41,3551–3554 (1970).

SUMMARY OF THE INVENTION

This invention relates to the treatment of a sample, such as a tissuesection from a plant or animal, with a compound or mixture of compoundsthat would perform multi-functional roles in the preparation of thesesamples for analysis, e.g., mass spectrometry or chromatography, atdesigned times determined by treatment conditions. These compounds wouldbe able to function as a surfactant or detergents in helping solubilizehydrophobic or other non-soluble compounds. Due to built in cleavablebonds, appropriate treatment of the sample, for example, with acid,base, heat, light, etc., would then cause decomposition of the agent totwo or more smaller parts, each of which does not materially interferewith the analysis. Further, each part may in itself perform a furtherfunction; for example, one part may help solubilize compounds present inthe mixture and the other, for MALDI mass spectrometry, may formcrystals in the same way matrix acts in common MALDI analysis.

Accordingly, the present invention provides, in part, compositions andmethods including, but not limited to: novel cleavable surfactants andmethods for preparing cleavable surfactants and using them in proteomicanalysis including for matrix assisted laser desorption ionization massspectrometry (MALDI MS). Certain compositions disclosed herein includethe surprising properties of being a surfactant that yields one or moreanalyte assisting molecules upon cleavage including a MALDI matrixcomposition and a volatile solvent. No aspect or embodiment of thepresent invention including any claim is bound by theory or mechanism.

In embodiments of the present invention, compounds of the presentinvention may be constructed or synthesized in two parts, connected by alinking group that can be cleaved by the addition of another chemicalagent or energy source. The portion of the compound that would act as amatrix after cleavage would be polar in nature and, in certainembodiments, be a cinnamic acid analog or similar compound. The secondpart of the molecule may be for example, a hydrophobic molecule such ashexane or octane alkyl group with a functional group such as thiol oralcohol. After cleavage, this compound may act as a solvent, allowingsolubilization of other compounds present including the other part ofthe agent. The linkage between the parts may comprise a bold such as,for example, a disulfide, thio ester, etc. that would preferably bestable until exposed to a chemical or energy source whereby it wouldcleave into the two parts described above.

Advantages described in certain aspects and embodiments of the presentinvention include that hydrophobic elements, such as certain polymers,polypeptides, proteins, and components of cell samples, and tissuesamples, etc. can be isolated and extracted using a detergent orsurfactant and then the surfactant compound is treatable or treated toyield cleavage compositions with different and useful properties. Forexample, certain novel surfactant compositions described herein losetheir surfactant properties upon cleavage of a linker group and thecleavage products are easily removed from the sample especially incomparison to the parent compounds or other surfactants which tend tostick to hydrophobic molecules.

One aspect of the present invention comprises surfactants including aMALDI MS matrix joined to a hydrophobic tail group by a cleavablelinker.

Another aspect of the present invention provides cleavable surfactantshaving a cinnamic group joined to a hydrophobic tail group by acleavable linker. Still another aspect of the invention provides asinapinic group and a hydrophobic group joined by a cleavable linker. Incertain embodiments, the linker comprises a disulfide group, a thioestergroup, or a ketal group. In certain preferred embodiments, the linker isa thioester group.

Another aspect of the present invention provides novel cleavablesurfactants having a polar head group joined to a hydrophobic tail by atleast one cleavable linker.

Another aspect of the invention provides certain novel cleavablesurfactants which lose their surfactant properties upon a cleavage.

Still another aspect of the present invention provides methods for usingsurfactants (novel to this invention or otherwise) for analysis ofmolecules, proteins, polypeptides, polymers and the like that arehydrophobic or include hydrophobic regions or domains.

In certain embodiments, methods are provided herein for using novelsurfactants of the present invention in the preparation of biologicalsamples or polymers for mass spectral analysis and preferably MALDI MSanalysis. Advantages of these methods over the prior art is that thesurfactants can be cleaved to yield a sample with analyte useful forMALDI MS analysis.

In certain preferred embodiments, methods are provided herein fortreating tissue specimens or cell samples (eg., for preparation orisolation of hydrophobic proteins or other molecules.

The processes of the present invention may include a enzymatic digestionof the hydrophobic protein before or after cleavage of the detergent. Inthis embodiment, the fragments may then be subjected to MS/MS forsequence analysis and identified using database searching.

Although certain aspects, embodiments, drawings and elements of theinvention are described herein, these are meant to be illustrative andnot limiting. For example, one of ordinary skill in the art will be ableto establish equivalents to certain elements herein, these equivalentsare considered to be within the spirit and scope of the presentinvention.

The cleavable detergents/surfactants of the present invention have beenfound to increase the signal intensity of high molecular weight proteinsin MALDI analyses, and help eliminate the suppressive effects ofdetergents in MALDI-MS. Additionally, the detergents/surfactants of thepresent invention increase the number of ions detected in mouse livertissue extracts, yielding a more complete peptide/protein profile.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference is now made to the accompanying drawings, which form a part ofthe specification of the present invention.

FIG. 1: a chart describing the MALDI mass spectra of a cleavabledetergent of the present invention in an analysis of mouse liver.

FIG. 2: a chart describing the MALDI mass spectra of a cleavabledetergent of the present invention in an analysis of mouse liverextract—high mass.

FIG. 3: a chart describing the MALDI mass spectra of a cleavabledetergent of the present invention in a direct analysis of mouse liver.

FIG. 4: a chart describing mass spectrometry analysis using analpha-cyano detergent of the present invention.

FIG. 5: a chart describing mass spectrometry analysis using analpha-cyano detergent of the present invention treating E. coli.

FIG. 6: a chart describing mass spectrometry analysis of mouse liverusing an alpha-cyano detergent of the present invention.

FIG. 7: a chart describing mass spectrometry analysis of direct tissueusing an alpha-cyano detergent of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves problems in the prior art associated withanalysis of hydrophobic molecules. As indicated, many analytical systemsfunction best when samples are aqueous or the molecules being analyzedin the sample are solubilized in an aqueous environment. For example,mass spectrometry (MS), and particularly matrix assisted laserdesorption ionization MS (MALDI MS) is a powerful analytical toolcapable of resolving or discriminating between molecules within one or afew atomic units of mass. MS is also exquisitely sensitive with possibledetection capabilities in the picomole or even femtomole range.

MS analysis of hydrophobic molecules or molecules with significanthydrophobic regions has proven troublesome. These molecules aredifficult, or sometimes essentially impossible, to suspend in aqueoussolution. They tend to aggregate and precipitate out of solution as thehydrophobic domains interact in a manner to minimize contact with theaqueous environment of typical MS samples preparations.

Molecules of special commercial importance include hydrophobic polymers,such as certain constituents of plastics; hydrophobic polypeptides, forexample membrane associated proteins, receptors; and lipids, lipophilliccellular components, and hydrophilic extracellular components. Thetypical approach to manipulating such molecules is to apply detergentsor surfactants to bring the hydrophobic molecule of interest out of itsnative environment and into a more aqueous environment. Surfactantsgenerally include a hydrophilic (or polar) head group and a hydrophobictail. They may arrange about a hydrophobic molecule with the tailsinteracting with hydrophobic areas on the molecule and the polar headgroup interacting with water in the environment.

For example, receptor proteins are often associated with or insertedinto the plasma membrane of a cell and are generally hydrophobic innature (at least the lipid associated portions thereof). Surfactants areuseful to isolate the receptor protein away from the plasma membrane.However, surfactants are also notorious for disrupting MALDI MSanalysis. The addition of common surfactants such as sodium dodecylsulfate, triton X, and tween essentially eliminates a molecular signalgenerated by MALDI MS as well as electrospray MS.

The present invention provides compositions and methods that solvesthese and other problems of the prior art.

1.0 Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Descriptions ofpreferred methods and compositions are provided herein, but should notbe construed to be limiting. No aspect, embodiment, or element of thepresent invention, including the claims, is limited or bound by theoryor mechanism of operation.

The terms “peptide”, “polypeptide”, and “protein” are usedinterchangeably herein unless a higher order conformation of apolypeptide is stated to be important, then “protein” may indicate thehigher order structure while “polypeptide” refers to the amino acidsequence.

The meaning of hydrophobic molecules, including synthetic and naturalpolymers, is known in the art. When referring to a hydrophobic protein,it is understood that the protein may have a “net” hydrophobicity, thisis, overall the protein is more hydrophobic than hydrophilic. Nethydrophobicity is determined using a hydropathic index of amino acids.For example, each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In this example, the more positive values are more hydrophobic.(For example, see Kyte et al., J. Mol. Biol. (1982) 157(1):105–32,incorporated herein by reference)

Hydrophobic proteins are those that have a positive total hydropathicindex after the following operation: each amino acid in the polypeptidechain is converted to its respective index value and the values aresummed to yield a total hydropathic index. Thehydrophobic/non-hydrophobic nature of polypeptides and peptides canlikewise be determined. It is understood that certain proteins andpolypeptides may have regions that are hydrophobic and that theseregions interfere with analysis or usefulness of the molecules, forexample, MALDI MS. In these cases, the hydropathic index for the regionis of interest and is determined. In certain cases, the region willcomprise consecutive amino acids and in other cases the region willcomprise a hydrophobic surface brought together by higher order foldingof the polypeptide chain (such as, tertiary structure).

Hydrophobic also means “water fearing”, from the Greek wordshydro—“water” and phobo—“fear.” The hydrophobic effect is an entropydriven force that causes oil to separate from water. The hydrophobicforce is strong, though not typically as strong as covalent forces. Thisforce is one of the main determinants of the structure of globularprotein molecules, since the hydrophilic (water loving) parts of theprotein tend to surround the hydrophobic parts that cluster in thecenter, away from the aqueous (polar) solvent. In other proteins, thehydrophobic regions are exposed, but inserted into or associated withmembranes or other hydrophobic structures.

The meaning of a “polar head group” or “hydrophilic group” is known inthe art and generally means a group or molecule that is readily solublein an aqueous environment. The meaning of a “hydrophobic tail”,“hydrophobic tail group”, or “hydrophobic group” is known in the art andgenerally refers to a molecule that is not intrinsically soluble in anaqueous environment.

As used herein the terms “emulsifier”, “wetting agent”, “detergent”, and“surfactant” are used interchangeably to mean an agent that reduces asurface tension in water. For example, a surfactant promotes keeping ahydrophobic polypeptide or generally hydrophobic protein in an aqueoussolution.

2.0 Introductory Description of Certain Embodiments

Certain novel surfactants are described herein that include ahydrophilic or polar head group connected by one or more covalent bondsto a hydrophobic tail group by at least one cleavable linker Certainnovel surfactants of the present invention include a matrix head group,comprising a MALDI matrix, a MALDI matrix precursor, or (in certainembodiments) a derivative of a MALDI matrix. The matrix head group istypically a polar molecule or a polar molecule after cleavage of thecleavable liner. Detergents of the present invention include in certainembodiments, but without limit, zwitterionic detergent, anionicdetergent, cationic detergent and non-ionic detergent.

3.0 Cleavable Linkers

Any chemical group (one or more atoms) that combines a polar head groupwith a hydrophobic tail is contemplated to be useful in certainembodiments of the present invention. In certain embodiments, theinteraction between a linker and the head and tail groups can be ionicbonding, hydrogen bonding, and Van der Walls bonds. One or more covalentbonds is preferred. The present invention includes one or more linkers,one or more polar head groups, and one or more hydrophobic tail groups.In certain embodiments, a linker is any chemical group that combines amatrix head group with a hydrophobic tail including, without limit, byformation of any of the above mentioned bonds.

Preferred cleavable linkers include a ketal linkage, a disulfidelinkage, and a thioester linkage. In general, disulfide bond linkagesare cleaved by applying a reducing agent. For example, dithiothreitol(DTT), β-mercaptoethanol (BMT), hydrogen sulfide (H₂S), sodiumhydrosulfide (NaSH), acid (H⁺ in H₂O), or base (OH⁻ in H₂O); are usefulfor cleaving a disulfide linkers of the present invention. In addition,light energy (hυ), preferably in the ultraviolet range, is useful forcleaving a disulfide linker of the present invention. In general, ketallinkers are cleaved using acid (H⁺ in H₂O), or in certain embodiments,base (OH⁻ in H₂O). In general, a linker can be formed by synthesizing acinnamic molecule with an ester in a 1 position of the cinnamic ring anda nucleophilic group (e.g., —OH, or —NH₂ without limit) at the 2position. Either acid or base conditions can be used to cleave such alinker as the ester undergoes nucleophilic attack. In general, thioesterlinkers are cleaved using reducing agents, acid, or base (see above forexamples). In certain preferred embodiments, thioester linkages are usedto join a known MALDI matrix as the head group with a hydrophobic tail.This is because cleavage of the thioester linkage, in general, yields anunmodified matrix product along with the hydrophobic tail group (whichis generally an aliphatic alcohol). In additional embodiments, thioesterlinkages are used to join a suspected MALDI matrix or a derivative of aknown MALDI matrix

4.0 Cleavable Surfactants

The present invention provides novel surfactants useful for variousindustries including for manipulation and analysis of plastics andproteomics. In general, it is an object of the present invention thatthese surfactants are cleavable into non-surfactant or essentiallynon-surfactant components. (Although, the hydrophobic tail groups mightgenerally be considered to be weak surfactants by some in the art; thesedo not induce significant MALDI signal suppression and they havedistinct advantages over other, especially stronger, surfactants asdiscussed herein.) One advantage to the cleavable surfactants of thepresent invention is that the cleavage products are readily removed bystandard isolation techniques (e.g., dialysis, ion exchangechromatography, filtration); whereas, non-cleavable surfactants tend tostick to the protein, or other hydrophobic molecules and are difficultto remove from the sample without losing the analyte itself.

In certain preferred embodiments, the surfactant is made up of ahydrophobic group linked by a cleavable linker to a polar group, whereinthe polar group is a MALDI MS matrix or precursor thereof. Thus,cleavage of the surfactant results in the liberation or formation of aMALDI matrix, or a derivative of a MALDI matrix in the sample. Oneadvantage to this in certain embodiments, is that the surfactant used toisolate the hydrophobic molecule is cleaved to form the MALDI matrix.The surfactant properties of the parent detergent are lost and MALDI MSanalysis can be carried out without surfactant induced signalsuppression (or at least a reduction in signal suppression). Anadditional advantage is that the hydrophobic tail group is typicallychosen (see below) to have certain of the following properties: asolvent for the hydrophobic molecule, volatile which supports theformation of superior matrix crystals, and readily removable from thesample if desired (e.g., aliphatic groups such as hexane which generallyyields hexanol as synthesized herein or an aromatic such as a benzenewhich can be drawn off under vacuum).

5.0 Polar Head Groups

In certain general embodiments, the polar head group may be any compoundcompatible with being joined to the linker, is not a strong surfactant(as defined or determined by testing to see if MALDI signal suppressionis observed in the presence of the compound). In certain embodiments,preferred polar head groups comprise a MALDI matrix or a precursor orderivative thereof. In certain highly preferred embodiments, the polarhead group includes cinnamic acid, derivatives of cinnamic acid,sinapinic acid, alpha-cyano-4-hydroxycinnamic acid (CHCA), and2,5-dihydroxybenzoic acid (2,5-DHB). Examples of certain preferred polarhead groups useful for the present invention are described in Table 1,below. The table below list certain embodiments and is not intended tolimit the scope of the invention.

TABLE 1 Molecular Molecular Name Molecular Structure Formula Weightsinapinic acid (SA)(3,5-dimethoxy-4-hydroxycinnamic acid)

Formula I C₁₁H₁₂O₅ 225.22 alpha-cyano-4-hydroxycinnamic acid(CHCA)

Formula 2 C₁₀H₇NO₃ 190.18 gentisic acid (DHB)(2,5-dihydroxybenzoicacid)

Formula 3 C₇H₆O₄ 155.13 2′,4′,6′-trihydroxyacetophenone(THAP)

Formula 4 C₈H₈O₄ 186.17 3-hydroxypicolinicacid(HPA)(3-hydroxy-2-pyridinecarboxylic acid)

Formula 5 C₆H₅NO₃ 140.12 ditbranol (DIT)

Formula 6 C₁₄H₁₀O₃ 226.06 2,-(4-hydroxy-phenlyazo)-benzoic acid(HABA)

Formula 7 C₁₃H₁₀N₂O₃ 242.23 trans-3-indoleacrylicacid (IAA)

Formula 8 C₁₁H₉NO₂ 187.20 ferulic acid(4-hydroxy-3-methoxycinnamic acid)

Formula 9 C₁₀H₁₀O₄ 195.20 nicotinic acid-N-oxide

Formula 10 C₆H₅NO₃ 140.12 2′-6′dihydroxyacetophenone

Formula 11 C₈H₈O₃ 153.16 picolinic acid (PA)(2-pyridine carboxylicacid)

Formula 12 C₆H₅NO₂ 123.1 6-aza-2-thiothymine(ATT)

Formula 13 C₄H₅N₃OS 143.176.0 Hydrophobic Tail Groups

In certain general embodiments, the hydrophobic tail is any compoundcompatible with being joined to the linker, is not a strong surfactant(as defined or determined by testing to see if significant MALDI signalsuppression is observed in the presence of the compound). (In certainembodiments, no MALDI signal is obtainable with traditional surfactantsor surfactants (non-cleaved) of the present invention; thus,“significant” does not represent a high barrier in certain embodiments.)

In certain embodiments, the hydrophobic tail is an aromatic. In certainpreferred embodiments, the hydrophobic group is an aliphatic group with2–20 carbons. In certain, highly preferred embodiments, the hydrophobicgroup is an aliphatic group with 4 to 8 carbons. In certain, preferred,the hydrophobic group is an aliphatic group with 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons.

7.0 General Structure of Cleavable Compounds of the Present Invention

The cleavable detergents/surfactants of the present invention includethose of the following basic formula:

In preferred embodiments, the hydrophobic tail may comprise alkyl,alkenyl, alkynyl groups containing 2–20 carbons. Preferably these groupscomprise straight chain or branched hydrocarbons, and/or single ormultiple chain hydrocarbons. Preferably, the length is 4–12 carbons.Most preferably, the carbon chains have 6 or about 6 carbon atoms.

The cleavable linker may be acid cleavable. Preferred acid cleavablelinkers include acetal/ketal linkers such as:

where R₁ is independently —H or —(CH₂)₀₋₁₉CH₃.

The cleavable linker may also be fluoride cleavable. Fluoride-cleavablelinkers may include:

R₁ is independently —CH₃,

The cleavable linker may also be a disulfide/thioester such as:

Tail-S—S-Head

Furthermore, the cleavable linker may also be photocleavable. An exampleof a photocleavable linker of the present invention includes:

X is a group NH₂ or OH. R₁ is —H, —CH₃, —F, —Cl, —Br, —I, or, —CN. Headgroups are attached independently on each of C4, C5, and C6.

The polar head may be the polar head in conventional cleavabledetergents/surfactants, including cationic, anionic, Zwitterionic,non-ionic carbohydrates, and MALDI matrices. Examples include thefollowing:

Cationic

N⁺[(CH)₀₋₃CH₃]₃, P⁺[(CH)₀₋₃CH₃]₃.

Anionic

SO₃ ⁻, SO₄ ⁻, CO₂ ⁻, PO₄ ⁻,

Zwitterionic

(In an all the above polar head groups, n is an integer from 1–12,preferably from 1–6.)

Amino Acids, including:

Cystine

Including cystine containing peptides. (6 amino acids or less).

Non-Ionic Carbohydrates, including:

Including polysaccharides. (3 carbohydrates or less).

n is an integer from 1 to 20.

MALDI Matrices, including:

In other perferred embodiments of the present invention, the cleavablecompounds of the present invention may have more than one cleavablelinker, and include compounds of the following general formula:

In this embodiment, the linkers, tails, and polar heads described abovemay be used. Additionally, it is preferred that the MALDI matrix isbased on the following compounds:

8.0 Isolation of Hydrophobic Proteins/Polypeptides

In certain embodiments, membrane bound proteins are liberated from asample of cells (cultured or collected tissue), extracted or isolatedusing standard procedures except that the surfactant utilized is acleavable surfactant, preferably one described herein and morepreferably a matrix cleavable surfactant. The cleavable surfactant iscleaved (e.g., by acid) and the sample is analyzed by MALDI MS.

In certain embodiments, membrane bound proteins are liberated from asample of cells (cultured or collected tissue), extracted or isolatedusing standard procedures including that a standard surfactant isutilized (e.g., SDS or triton X). The standard surfactant is exchangedwith one of the present invention (e.g. by dialysis exchange) and thesample is collected The cleavable surfactant is cleaved (e.g., by acid)and the sample is analyzed by MALDI MS.

9.0 MALDI MS Analysis of Tissue Sections

In certain embodiments, a tissue section is obtained. The section istreated with a cleavable surfactant, preferably one described herein andmore preferably a matrix cleavable surfactant. The section is incubatedto allow certain of the proteins and other hydrophobic molecules tobecome solubilized by the cleavable surfactant. The cleavable surfactantis cleaved (e.g., by acid or reducing agent) and the tissue section isanalyzed by MALDI MS.

In an additional embodiment of the present invention, compounds of thepresent invention may be used in one dimensional and two dimensionalpolyacrylamide gel electrophoresis. Two dimensional polyacrylamide gelelectrophoresis (2D-PAGE) is a technique commonly used for the analysisof mixtures of proteins. (U. K Laemmli, Nature 227, 680–685, 1970).Proteins are separated first by an electrophoretic such as isoelectricfocusing followed by a second dimension separation based on proteinsize. Sodium dodecyl sulfate, the detergent most often used with2D-PAGE, forms stable non-covalent complexes with proteins. The SDScomplexed proteins have identical charge density; therefore, theyseparate in an electrical field according to their size. This techniqueis capable of separating a complex protein mixture into several hundredindividual components that can be excised from the gel and furtheridentified by other techniques. One such technique is mass spectrometry.The direct analysis of proteins removed from electrophoresis gels isoften difficult Commonly, the samples contain detergent concentrationsthat hinder analysis by mass spectrometry. The direct analysis ofproteins removed from electrophoresis gels is often difficult. Commonly,the samples contain detergent concentrations that hinder analysis bymass spectrometry. In MALDI analysis for example, this problem is theresult of the tendency of the detergent to aggregate or associate withthe protein preventing proper incorporation into the matrix crystal.Special steps must be taken to remove the interference prior to analysisby MALDI MS. Examples of such measures include, but are not limited toelectroblotting of PAGE gels and detergent exchange of SDS with a moreMALDI tolerant detergent like n-octyl-glucoside, for example.

An alternative approach is to use cleavable analogs to commonly useddetergents in SDS-PAGE. For example, anionic analogs to SDS of thepresent invention such as the following:

Detergents of the present invention that may be used include anycationic or anionic cleavable detergent. Preferably, anionic cleavabledetergents are used.

Zwitterionic or non-ionic detergents of the present invention may beused for simple 1D gel electrophoresis in which proteins are separatedbased on isoelectric point. Example of preferred embodiments include thefollowing:

These compounds are applied according to established protocols in theanalysis of proteins by gel electrophoresis. Subsequent analysis of theseparated biomolecules is accomplished by excising the proteins from thegel, reconstituting the protein, and applying the sample to a MALDItarget. The appropriate cleavage agent is applied to the sample alongwith the matrix, if necessary, allowing more accurate mass spectrometrydetermination of molecular weight. With respect to electrophoresis ofproteins, see Westermeier, Electrophoresis in paractice, 3^(rd) Edition,2001; and Hames, Gel electrophoresis of proteins: a practical approach,3^(rd) Edition, 1998.

The following examples are for illustrative purposes, and not intendedto limit the scope of the invention as defined by the claims.Additionally, in practicing the present invention, one of ordinary skillin the art would understand that various modifications to the followingprocedures would be routine, in light of the teachings herein, and thatsuch modifications would be within the spirit and scope of the presentinvention.

EXAMPLES Example 1

Example 1 is a selection of embodiments of cleavable detergents orsurfactants of the present invention, including the hydrophobic tail,cleavable linker, and polar head group.

A composition of Formula 14:

or a salt thereof, wherein:

-   -   R1 and R2 is each independently —H, —OCH₃, —(CH₂)₁₋₆CH₃, or        —O(CH₂)₁₋₆CH₃;    -   R3 and R4 is each independently —H, —OCH₃, —OH, —NH₂,        —(CH₂)₁₋₆CH₃, or —O(CH₂)₁₋₆CH₃;    -   R5 and R6 are each independently —(CH₂)₁₋₁₉CH₃;    -   R7 is independently —(CH₂)₁₋₁₉CH₃; and    -   R8 is independently —(CH₂)₁₋₆,    -   X is independently SO₃ ⁻, SO₄ ⁻, or NH₃ ⁺.

A preferred embodiment of formula 14 is where that R1 and R4 are H, andR7 is methyl. The basic structure defined by R5—O—C—O—R6 is that of aketal linkage. The present set of structures is especially useful forthe ability to degrade the surfactant by cleavage at the ketal yieldingmolecules with reduced MALDI signal suppression.

Compositions of Formula 15:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   R2, R3, R4 is each independently —H, —OCH₃, —(CH₂)₁₋₆CH₃, or        —O(CH₂)₁₋₆CH₃;

Compositions of Formula 16:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   R2 is —H, methyl, halide, halogen, or cyano (—CN); and    -   R3 independently —H, —OH, —OCH₃, —(CH₂)₁₋₆CH₃, or —O(CH₂)₁₋₆CH₃;

Compositions of Formula 17:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   R2 is —H, methyl, halide, or cyano (—CN);    -   R3, R4, R5, and R6 is each independently —H, —OCH₃,        —(CH₂)₁₋₆CH₃, or —O(CH₂)₁₋₆CH₃; and    -   X is oxygen, —NH, or a nucleophile.

Compositions of Formula 18:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   R2 is independently —(CH₂)₁₋₆; and    -   R3, R4, R5, and R6 is each independently —H, —OCH₃,        —(CH₂)₁₋₆CH₃, or —O(CH₂)₁₋₆CH₃. Additionally, the chain with R1        and R4 are substituted one for the other.

Compositions of Formula 19:

or a salt thereof, wherein:

-   -   n is an integer of from 1 to 20;    -   Y is independently positioned at one or more of C3, C4, C5, or        C6, wherein Y represents independently —H, or a straight or        branched chain, substituted or unsubstituted: alkyl, alkene, and        alkyne; and    -   X is oxygen, —NH, or a nucleophile.

Included in this Example are the following formulas:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   Y is independently positioned at one or more of C3, C4, C5, or        C6, wherein Y represents independently —H, —OCH₃, —(CH₂)₁₋₆CH₃,        or —O(CH₂)₁₋₆CH₃; and    -   X is oxygen, —NH, or a nucleophile.

Also included in this Example are the following formulas:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   Y is independently positioned at one or more of C3, C4, C5, or        C6, wherein Y represents independently —H, —OCH₃; —(CH₂)₁₋₆CH₃,        or —O(CH₂)₁₋₆CH₃; and    -   X is oxygen, —NH, or a nucleophile.

Also included in this Example are the following formulas:

or a salt thereof, wherein:

-   -   R1 is independently an aromatic or —(CH₂)₁₋₁₉CH₃;    -   Y is independently positioned at one or more of C3, C4, C5, or        C6, wherein Y represents independently —H, —OCH₃, —(CH₂)₁₋₆CH₃,        or —O(CH₂)₁₋₆CH₃; and    -   X is oxygen, —NH, or a nucleophile.

Finally, also included in this Example are the following formulas:

Example 2

This Example describes the synthesis and cleavage ofalpha-cyano-4-hydroxy-cinnamic acid detergent, a preferred embodiment ofthe present invention.

Synthesis of Chloromethyl ether: A volume of 5 mL (39.4 mmol) ofchlorotrimethylsilane (TMS-Cl) and 0.3 g of paraformaldehyde were placedin a flame dried 25 mL round bottom flask. The reagents are allowed tostir under inert atmosphere until homogeneous. A volume of 2.27 mL (10mmol) of n-dodecanol were added drop wise to the reaction vessel. Thereagents react at room temperature for a period of two hours. The TMS-Clis removed under vacuum followed by a vacuum distillation of theproduct, chloromethyl ether. A total of 0.973 g (41% yield) of productwere collected at 106°–109° C. at 0.4 torr.

Synthesis of the methoxyalkyl ether of α-cyano-4-hydroxycinnamic acid:Powdered NaOH (107 mg, 2.68 mmol) was dissolved in 2 mL ofdimethylsulfoxide in a flame dried 25 mL round bottom flask. To thismixture, 0.302 g (1.35 mmol) of α-cyano-4-hydroxycinnamic acid was addedto the reaction. The reaction mixture was placed under inert atmosphereand allowed to stir until all reagents were dissolved. At this time,0.255 g (1.08 mmol) of the newly synthesized chloromethyl ether wasadded drop wise to the reaction mixture. The reaction was allowed tostir for a period of 12–16 hours. TLC confirmed that the reaction wascomplete. Reaction mixture was diluted with chloroform and washedrepeatedly with saturated NaCl. Remaining traces of DMSO were removedunder vacuum with an in-line cold trap. A weight of 0.653 g (65.3%yield) of product was purified.

Example 3

This Example describes the synthesis and cleavage of sinapinic aciddetergent, a preferred embodiment of the present invention.

Synthesis of Chloromethyl ether: A volume of 5 mL (39.4 mmol) ofchlorotrimethylsilane (TMS-Cl) and 0.3 g of paraformaldehyde were placedin a flame dried 25 mL round bottom flask. The reagents are allowed tostir under inert atmosphere until homogeneous. A volume of 2.27 mL (10mmol) of n-dodecanol were added drop wise to the reaction vessel. Thereagents react at room temperature for a period of two hours. The TMS-Clis removed under vacuum followed by a vacuum distillation of theproduct, chloromethyl ether. A total of 0.973 g (41% yield) of productwere collected at 106°–109° C. at 0.4 torr.

Synthesis of Protected Sinapinic Acid (5):

-   -   Synthesis of trimethylsilylethyl bromoacetate (3): In a dry 50        mL round bottom flask, 1.65 mL (11.54 mmol) of trimethylsilyl        ethanol (1), 0.88 mL (11 mmol) of pyridine, 0.124 g (1 mmol) of        N,N-dimethylaminopyridine, and 20 mL of methylene chloride were        placed. The reaction mixture was placed under inert atmosphere.        A volume of 0.97 mL bromoacetyl bromide (2) was added drop wise        to the reaction mixture. The reaction proceeded for two hours at        room temperature. Reaction was washed twice with 1 M HCl        followed by a wash with saturated NaCl. The organic layer was        dried over MgSO₄. A quantitative yield was obtained, 2.74 g of        trimethylsilylethyl bromoacetate (3).    -   Synthesis of phosphonium salt (4): To the 2.74 g (11.46 mmol) of        trimethylsilylethyl bromoacetate (3) previously synthesized,        4.51 g (17.20 mmol) of triphenylphosphine and 20 mL of ethyl        acetate were added. The reagents were stirred at room        temperature for 24 hours. A white precipitate formed which was        isolated by vacuum filtration. A mass of 4.08 g (8.14 mmol,        71.0% yield) of phosphonium salt (4) was isolated.    -   Synthesis of protected sinapinic acid (5): An amount of 0.824 g        (1.65 mmol) of the phosphonium salt (4) previously synthesized        was added to 0.23 mL (1.65 mmol) of trimethylamine in 5 mL of        benzene. A yellow solution of ylide formed within 30 minutes.        Syringaldehyde (0.274 g, 1.5 mmol) was added to the ylide and        stirred for 16 hours. The organic layer was washed with 1 M HCl.        Further purification was accomplished using flash chromatography        (3:2 ethyl acetate/hexane). A yield of 73.1% (0.356 g) of        protected sinapinic acid was obtained.

Synthesis of the methoxakyl ether of sinapinic acid: In a dry roundbottom flask, 0.2 mL (1.43 mmol) of trimethylamine, 0.308 g (0.951 mmol)of protected sinapinic acid (5) and 0.271 g (0.836 mmol) of the newlysynthesized chloromethyl ether were added placed. The reaction wasstirred for 12 hours at room temperature. Product 6 was purified usingalumina flash chromatography with methylene chloride as the mobilephase. An amount of 0.402 g (0.770 mmol, 92.1% yield) of compound 6 wasisolated.

Deprotection: To 0.402 g (0.770 mmol) of compound 6 in 2 mL oftetrahydrofuran, 0.65 g of tetrabutylammonium fluoride was added. Animmediate yellow color was observed. The reaction proceeded at roomtemperature for one hour. The product was extracted from saturatedammonium chloride with methylene chloride. An amount of 0.320 g (0.762mmol, 99.0% yield) of deprotected sinapinic acid detergent was obtained.

Example 4

This Example describes the systhesis of dihydroxybenzoic acid detergent,and acid and/or fluoride cleavage.

Example 5

This Example describes the synthesis and cleavage of disulfidedetergent, a preferred embodiment of the present invention.

Octylthiophthalimide was prepared according to behforouz et al.: avolume of 10 ml (57.62 mmol) of octane thiol were placed in a 250 mlround bottom flask with 75 ml of heptane. Chlorine gas was bubbledthrough the solution. Conversion of octane thiol to the correspondingsulfenyl chloride, as monitored using gas chromatography, occurred inapproximately 30 minutes. Drop wise addition of 8.5 g (57.8 mmol) ofphthalimide and 8 ml (57.5 mmol) of trimethylamine in 75 ml ofn,n-dimethylformamide converted the sulfenyl chloride tooctylthiophthalimide. After stirring 30 minutes, the reaction was addedto 100 ml of cold water, then the precipitant was collected byfiltration. The product was further purified using column chromatography(1:2 ethyl acetate/hexanes) yields greater than 95% conversion of octanethiol were obtained.

Synthesis of glucose based non-ionic disulfide detergent according toHarpp et al.: Equimolar amounts of 1-thio-β-D-glucose andoctylthiophthalimide were refluxed in ethanol for 12 to 20 hours.Formation of the unsymmetrical disulfide proceeded with greater than 80%yield.

Example 6

This Example describes fluoride cleavable detergents of the presentinvention.

R₁=Akyl, Alkenyl, Alkynyl (C4–C20).

-   -   R₂=-Me, -tBu, -φ.    -   X=Cl, Br, I

Example 7

This Example describes synthesis of fluoride clevable analogs of CTAB.

Synthesis of ethyl alkyldimethylsilylacetate (3): All reagents werepurified prior to use. A dry 250 mL flask was fitted with a condenserand an addition funnel. An amount of 2.03 g (31.0 mmol) of powdered zincwas placed into the flask under inert atmosphere. To a mixture of 65 mLbenzene and 15 mL diethyl ether, 3.46 g (16.77 mmol) ofchlorodimethloctyl silane (1) and 2.4 mL (21.6 mmol) of ethylbromoacetate (2) were added and the reagents were placed in the droppingfunnel. Approximately 10 mL of the reagent mixture were added to thezinc; initiation was evident after approximately 5 minutes. Theadditional reagents were added drop wise over 30 minutes. The reactionwas allowed to proceed for 20 hours at room temperature. The reactionwas quenched using 40 mL of 1 M HCl. The organic layer was furtherwashed with 1 M HCl, water, saturated bicarbonate, and water. Theorganic layer is dried over magnesium sulfate. Compound 3 was producedin 82.5% yield (3.57 g).

Synthesis of alkyldimethylsilyl ethanol (4): An amount of 2.05 g (7.75mmol) of compound 3 was refluxed with 0.55 g (14.3 mmol) of lithiumaluminum hydride in 50 mL of ether for one hour. After cooling to roomtemperature, 0.55 mL of water, 0.55 mL of 15% NaOH, and 1.65 mL of waterwere added sequentially with stirring. The precipitates were removed byfiltration through celite. The alcohol was produced at 85.7% yield (1.47g).

Synthesis of alkyldimethylsilylethyl bromoacetate (6): A volume of 0.658mL (3.05 mmol) of alkyldimethylsilyl ethanol (4), 0.267 mL (3.3 mmol) ofpyridine, 0.122 g (1 mmol) of N,N-dimethylaminopyridine, and 20 mL ofmethylene chloride were placed in a 50 mL round bottom flask. Thereaction mixture was placed under inert atmosphere. A volume of 0.290 mLbromoacetyl bromide (5) was added drop wise to the reaction mixture. Thereaction proceeded for two hours at room temperature. The reactionmixture was washed twice with 1 M HCl followed by a wash with saturatedNaCl. The organic layer was dried over MgSO₄. Quantitative yield wasobtained, 1.01 g of alkyldimethylsilylethyl bromoacetate (6).

Synthesis of fluoride cleavable cationic detergent (8): Trimethyl amine(7) was condensed over 0.88 g of compound (6) in a pressure tube. Tubewas sealed, and the reaction stirred overnight at room temperature. Abrown precipitant formed within an hour. The tube is cooled to −78° C.,opened, then the reaction is allowed to return to room temperature. Whenthe trimethyl amine had evaporated, 0.89 g (2.24 mmol) of product (8)remained (86.1%).

Example 8

This Example describes synthesis and cleavage of fluoride cleavabledetergents with matrix headgroups.

Example 9

This Example describes synthesis of sinapinic acid detergents that arefluoride cleavable.

Example 10

This Example describes the synthesis and cleavage of m-PPS, a preferredembodiment of the present invention.

FIG. 1 describes the MALDI mass spectra of a compound of this example.The solvent composition is about 25 mM in ACN/water. The ratio oftissue/solvent required is about 50 mg/mL. The concentration of acid isabout 1:10 ratio of 1% HCl/detergent. The mouse liver was homogenized inthe appropriate detergent solution and centrifuged for 10 min. Thecleavage was initiated on plate by 0.1 uL 1% HCl to 0.5 uL drop of liverextract. 0.5 uL of sinapinic acid (10 mg/mL in 50% CAN) was added after2–3 minutes. The graphs of FIGS. 2 and 3 were generated as part of thisExample as well.

Example 11

This Example describes mass spectrometry experiments conducted accordingto the present invention where a solution of AC detergent was preparedin 10% acetonitrile. A standard mixture of peptides was used. Cleavagewas carried out on target using 1:10 ratio of 1% HCl/detergent solution.FIGS. 4–7 were generated as part of this Example.

It would be obvious to one of ordinary skill in the art that the presentinvention may be practiced using equivalents of the embodimentsdescribed herein. Such equivalents are intended to be encompassed by theclaims of the present invention.

All patents and publications cited herein are hereby expresslyincorporated by reference in their entirety.

1. A compound of the following formula:

wherein n is an integer from 1 to 12, R is —(CH₂)₀₋₁₉CH₃, and R₁, is—CH₃,


2. The compound of claim 1, of the following formula:

wherein n is an integer from 1 to 12, R is —(CH₂)₀₋₁₉CH₃, and R₁ is—CH₃,


3. The compound of claim 1, of the following formula:

wherein R₁ is —CH₃,


4. The compound of claim 1, of the following formula:

wherein R₁ is —CH₃,

and n is an integer from 1 to
 12. 5. The compound of claim 1, of thefollowing formula:


6. A method for isolating a hydrophobic molecule, comprising: providinga plasma that has a hydrophobic molecule; applying a cleavablesurfactant/detergent compound of the following formula:

 wherein n is an integer from 1 to 12, R is —(CH₂)₀₋₁₉CH₃, and R₁ is—CH₃,

cleaving the surfactant from the hydrophobic molecule; and analyzingsaid hydrophobic molecule with mass spectrometry.
 7. The method of claim6, wherein the cleavable surfactant/detergent compound is of thefollowing formula:

wherein n is an integer from 1 to 12, R is —(CH₂)₀₋₁₉CH₃, and R₁ is—CH₃,


8. The method of claim 6, wherein the cleavable surfactant/detergentcompound is of the following formula:

wherein R₁ is —CH₃,


9. The method of claim 6, wherein the cleavable surfactant/detergentcompound is of the following formula:

wherein R₁ is —CH₃,


10. The method of claim 6, wherein the cleavable surfactant/detergentcompound is of the following formula: